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Chapter 5
TABLE OF CONTENTS
Biochemical Oxygen Demand (BOD)
Page
Section 1: General
...


2

Section 3: Glossary
...


3

Section 5: Safety and Hygiene
...


4-5

Quiz 5
...


5

Section 7: BOD - Description of Method
...


5-7

Section 9: Determination of Sample Size
...


8

Quiz 5
...


9

Section 11: Pretreatment of Sample
...
3
...
11-12
Section 13: Calculations
...
12-13
Section 15: Precision and Accuracy
...
4
...
14-17
Quiz 5
...


18

Section 17: QA/QC
...
19-21
Appendix A: References
Appendix B: Preparation of Chemicals
Appendix C: Sample Bench Sheet
Appendix D: Methods Checklist BOD
Appendix E: BOD Survivors Guide
Appendix F: Bench Sheet

Chapter 5 - 1

Chapter 5
BIOCHEMICAL OXYGEN DEMAND (BOD)
Section 1: GENERAL
In the presence of free oxygen, aerobic bacteria use the organic matter found in wastewater as “food”
...
The more “food” present in the waste,
the more Dissolved Oxygen (DO) will be required
...

Section 2: BOD INTRODUCTION
The BOD test is used to measure waste loads to treatment plants, determine plant efficiency (in terms of
BOD removal), and control plant processes
...
A major disadvantage of the BOD test is the amount of time (5 days) required to obtain
the results
...

Because the test is performed over a five day period, it is often referred to as a “Five Day BOD”, or a
BOD5
...
These organisms can exert an oxygen demand as
they convert nitrogenous compounds (ammonia and organic nitrogen) to more stable forms (nitrites and
nitrates)
...

Sometimes it is advantageous to measure just the oxygen demand exerted by organic (carbonaceous)
compounds, excluding the oxygen demand exerted by the nitrogenous compounds
...
The result is termed “Carbonaceous Biochemical Oxygen Demand”, or CBOD
...

Anaerobic: A condition in which “free” or dissolved oxygen is not present in an aquatic environment
...


Chapter 5 - 2

Nitrification: An aerobic process in which bacteria change ammonia and organic nitrogen in wastewater
into oxidized nitrogen (usually nitrate)
...
(The first stage is called the “carbonaceous stage”
...

Respiration: The process in which an organism uses oxygen for its life processes and gives off carbon
dioxide
...


Section 4: APPROVED METHODS
Always refer to your facility MPDES Permit and 40 CFR Part 136 for the approved sampling and test
procedure
...


Section 5: SAFETY AND HYGIENE
When testing for BOD, the concerns for safety involve wastewater hazards and exposure to chemicals
...


Cover all abrasions and wear good quality latex gloves when in direct contact with raw wastewater
...


Wash hands frequently, and always wash hands prior to eating or smoking
...


Clean up all spills of wastewater or buffers immediately
...


Wear a protective smock, apron, or lab coat, and surgical or rubber gloves when working in the
laboratory to protect clothes and skin
...


Read all labels carefully and know what to do in case of a spill
...


Always use a pipette bulb
...


Chapter 5 - 3

7
...
Mixing concentrated
acids or bases with water can create a significant amount of heat
...


Use care when handling sharps (broken glass etc
...
The sample must be representative,
collected properly, handled carefully and preserved correctly
...

TYPES OF SAMPLES
Samples used for the BOD test can be either grab or composite
...
The type and location of sample taken will depend on your facility NPDES Permit
...

Samples should be taken at a point where they will be well-mixed and proportional to the amount of the
flow
...
Testing should be started as
quickly as possible
...
Do not allow samples to freeze
...

NOTE: The 48 hours starts when the very first aliquot of a composite sample is collected (i
...
when the
composite sampler starts collecting a composite sample)
...
Sampling devices should be capable of collecting samples from well-mixed areas of tanks
and/or pipes, made of resistant materials that will not rust or corrode, capable of taking samples that are
proportional to the plant’s flow and easily cleaned (including acid cleaning)
...
Do not use containers such as coffee cans
...
These containers should have leak-proof caps or lids
...
This will prevent buildup (such
as grease and scum) from contaminating samples
...
This is especially important for containers used for
samples which are high in solid and/or grease content
...
One sampling container should not be used throughout the plant
...

Sample storage containers should also be cleaned thoroughly between samples
...
It is also
recommended that each sampling point have its own storage container
...
) The containers should be clean and dry before a new set of
grab or composite samples are stored in them
...
1
1
...


What can the BOD test be used for?

3
...
The DO content is determined
and recorded and the bottle is incubated in the dark for five days at 20°C
...
The decrease in DO is corrected for sample dilution, and represents the biochemical oxygen
demand of the sample
...
Test reagents are
as follows:
1
...


Magnesium sulfate solution (MgSO4 7H2O) *

Chapter 5 - 5

3
...


Ferric chloride solution (FeCl3) *

5
...


Sulfuric acid (H2SO4), 1 N *

7
...
025 N

8
...


Acetic acid solution (CH3CO2H), (1+1) *

10
...
Starch indicator solution
12
...
Nitrification inhibitor (2-chloro-6-(trichloromethyl) pyridine) *
14
...

NOTE: Use only high-grade distilled or deionized water
...
01 mg/L
copper, and be free of chlorine, chloramines, caustic alkalinity, organic material, or acids
...

EQUIPMENT

1
...


300 mL BOD bottles

3
...


250 mL graduated cylinders

5
...


25 mL measuring pipettes (wide-mouth)

7
...


100 mL beaker

9
...
250 mL Erlenmeyer flask
11
...
1 mL
12
...
Pipette bulb

Chapter 5 - 6

14
...
Magnetic stirrer and stirring bars **
** Optional equipment
Section 9: DETERMINATION OF SAMPLE SIZE
The BOD test relies on a measurable depletion of DO over a specified period of time
...
This dilution is done by adding dilution water to
the sample in the BOD bottle
...
If the final DO is too low, the
BOD cannot be determined
...

One of the most difficult steps in the BOD procedure is deciding how much sample to place in the BOD
bottles for incubation
...
In all cases, several different dilutions of each sample
should be prepared to obtain the desired DO depletions
...
The following
procedure can be used to calculate volumes for sample dilution from the estimated BOD
...
0 mg/L
...
0 mg/L and the residual DO at least 1
...


mL sample added to BOD bottle = (minimum allowable depletion, mg/L x Volume of BOD bottle,
mL)/estimated BOD, mg/L
Example:
minimum mL sample = [(8 mg/L - 6 mg/L) x 300 mL]/400 mg/L
minimum mL sample = (2 x 300)/400 = 600/400 = 1
...


mL sample added to BOD bottle = (maximum allowable depletion, mg/L x Volume of BOD bottle,
mL)/estimated
BOD, mg/L
Example:
maximum mL sample = [(8 mg/L - 1 mg/L) x 300 mL]/400 mg/L
maximum mL sample = (7 x 300)/400 = 2100/400 = 5
...
For the examples above, four bottles would be used with 1 mL, 3 mL, 4 mL, and 6 mL, and the
results averaged for the final BOD
...

Section 10: PREPARATION OF DILUTION WATER
It is very important that the distilled water used for dilution water be of high grade and free from
contaminants (such as copper and chlorine) which could inhibit the growth of bacteria
...
e
...

Prepare dilution water as follows:
1
...
This can be accomplished by
aerating with clean compressed air
...


2
...


3
...


NOTE: The Hach Company manufactures slurry pillows which can be used as a substitute for the nutrient
reagents
...

4
...


5
...
2 mg/L DO)
...


6
...
As an alternative, 3
...


Chapter 5 - 8

Quiz 5
...


What reagents are required for the BOD5 and/or CBOD5 test?

2
...


If the expected BOD of a sample is in the range 25 mg/L to 75 mg/L, what would be the
minimum and maximum sample volumes to use for the sample dilution?

Section 11: PRETREATMENT OF SAMPLE
Samples with extreme pH values and samples containing disinfectants such as residual chlorine must be
treated prior to testing
...
To
prevent this, samples which have pH values higher than pH 8
...
0 must be neutralized
to pH 7
...

NOTE: Neutralized samples must be seeded for the BOD test
...


Pour 50 mL of sample into a 100 mL beaker
...


Measure the pH of the sample using a pH meter
...
0 to pH 8
...


3
...
0
...


Calculate the amount of sulfuric acid or sodium hydroxide needed to neutralize 1000 mL of the
sample
...


Add the calculated amount of acid or base to the sample
...


Repeat steps 1-5 until the pH test shows pH 7
...


Chapter 5 - 9

Calculation
7
...
0 using the following formula:
mL needed = (mL acid or base used x mL total test sample)/mL sample portion used for
neutralization
...
3 mL of 1 N NaOH are used to neutralize 50 mL of sample to pH 7
...

Calculate the volume of NaOH to be added to neutralize the sample as follows:
mL 1 N NaOH needed = (1
...
Any samples containing residual chlorine must be pretreated to remove chlorine before the test is
run
...

NOTE: Those samples which are dechlorinated must be seeded for the BOD test
...


To a 250 mL Erlenmeyer flask, add 100 mL of a well-mixed portion of the sample to be
dechlorinated
...


Add 10 mL of either 1+1 acetic acid solution or 1+50 sulfuric acid solution to the flask and swirl to
mix
...


Add 10 mL of potassium iodide (KI) solution, and 1 mL of starch indicator solution
...


4
...


NOTE: Do not assume that the sample was not chlorinated simply because there is no reaction
...
The only way to be sure a sample is
not chlorinated is to know exactly where the sample was collected
...


If a blue color appears, titrate the treated portion of sample with 0
...
Record this amount on a lab sheet
...


Calculate the amount of sodium sulfite solution needed to dechlorinate the selected BOD sample
volume
...


Add the calculated volume of sodium sulfite to the BOD sample and mix thoroughly
...


Allow the sample to stand for 10 to 20 minutes, then repeat steps 1-3
...


If no chlorine is detected, continue with the BOD test procedure, otherwise continue with steps 5-8
until the sample is dechlorinated
...
Calculate the amount of sodium sulfite needed to dechlorinate the BOD sample using the following
formula:

Chapter 5 - 10

mL Na2SO3 needed = (mL Na2SO3 used x mL total test sample)/mL sample portion used for
dechlorination
...
0 mL of Na2SO3 is needed to titrate 100 mL of sample for dechlorination
...
0 mL x 1500 mL)/100 mL = 1500/100 = 15 mL

Quiz 5
...


Why must samples containing caustic alkalinity or acidity be adjusted before preparing
BOD dilutions?

2
...


Why must samples containing residual chlorine be dechlorinated before preparation of
BOD dilutions?

4
...


What must be done to samples which have been dechlorinated or adjusted for pH
variations?

Section 12: LABORATORY PROCEDURE
1
...


2
...
Add dilution water until the bottles are completely filled
...
If the meter method is used for DO measurements the initial and final DO determinations
can be performed on the same bottle
...
To inhibit the nitrifying
bacteria in the sample, add 3
...
Continue with the remaining procedural steps with both sets of
dilutions
...


Stopper each bottle taking care to avoid trapping air bubbles inside the bottles as the bottle stoppers
are inserted
...


Fill the top of each bottle neck around the stopper with dilution water
...


Determine the initial DO content on one of each set of duplicate bottles, including the dilution water
blank by one of the approved methods and record data on the lab sheet
...


Place the remaining bottles in the incubator at 20°C and incubate for five days
...


At the end of exactly five days (+/-3 hours), test the DO content of the incubated bottles
...


Calculate the BOD for each dilution
...
0 mg/L DO residual
...


9
...
If the quality of the
water is good and free from impurities, the depletion of DO should be less than 0
...
In any
event, do not use the depletion obtained as a blank correction
...
If nitrification inhibition is used, the BOD test must also be performed on a series of sample dilutions
which have not been inhibited
...
Report the results of the nitrification inhibited samples as CBOD5 and uninhibited samples as BOD5
...
2 mg/L
Final DO = 4
...
2 - 4
...
8 x 300)/5 = 1140/5 = 228 mg/L
Whenever a sample is dechlorinated, it must be seeded
...
A number of
BOD’s must be run on the seed material to determine the seed correction factor
...
These will include chlorine, caustic alkalinity or acidity,
mineral acids, and heavy metals (such as copper, zinc, chromium, and lead)
...
Growth of algae in the presence of light can
cause problems by actually increasing the DO of the sample before testing, which must be removed by
deaeration
...
To prevent this, all glassware should be acid cleaned on a regular basis
...
A 2% dilution
(6 mL per 300 mL BOD bottle) should yield 200 +/-37 mg/L BOD, after five days incubation at 20°C
...

The BOD test is a biological test, dependent on the actions of the microorganisms found in the wastewater
and, as such, is subject to a number of variations
...
Results can vary widely from day to day, or even hour to hour
...
This makes the BOD test a poor test for determining whether or not operational changes are
needed
...
For
example, some organic matter (like sugars or starches) are oxidized very easily and rapidly, and will
almost always result in measurable “BOD”
...
Although this is what actually happens in nature, it causes significant variation in BOD results
from sample to sample
...
For a highly nitrified effluent sample, the
difference can be as great as 50%
...
4
1
...


What is the accepted temperature range and time of incubation for the BOD test?

3
...


What are the criteria for most valid results of the BOD test?

Section 16: SEED CORRECTION PROCEDURE
The BOD test relies on the presence of healthy organisms
...
This process is known as seeding
...
There
are other techniques which can be used
...
) should be
performed prior to preparation of the sample dilutions for this test
...
This will help ensure
that the seed correction meets the 0
...

Place the material in a suitable container and incubate at 20°C for 24-36 hours
...
If
not, small quantities of digester supernatant, return activated sludge, or an acclimated seed material can
be used to increase the potency of the seed material used for the test
...
The seed correction should not exceed 1
...
The key to a good seed correction
is a relatively stable seed material which produces a good seed correction in every test situation
...

Prepare two bottles of each dilution for the seed control series
...
The percentage of seed used in
each dilution and the number of dilutions is optional, but sufficient dilutions should be used to ensure that
at least one dilution gives a depletion of 2
...
0 mg/L DO residual
...
At the end of
the incubation period, determine the final DO of the dilutions
...

#1 DO depletion = Initial DO - Final DO
Select the seed dilution(s) which meet the required criteria and calculate the BOD of the seed material
using formula #2 below
...
)
#2 Seed BOD = (DO depletion x 300)/Seed dilution, mL
The calculated seed BOD represents the BOD exerted by 300 mL of undiluted seed material
...

DETERMINATION OF SEED VOLUME
The most common methods for introducing the seed material into the sample dilutions are (a) addition of
the seed to the dilution water and (b) addition of the seed directly to the sample BOD bottles
...
Method (b) is somewhat easier to use as the
volume of seed for each dilution is constant
...

#3 Volume of seed in sample dilution = (Volume of seed in dilution water x dilution water in
sample, mL)/Total volume of dilution water
...
The seed correction is actually the oxygen demand exerted by the oxidation of the
small amount of organic matter in the seed material in the sample dilutions
...

#4 Seed Correction = (Seed BOD x mL seed in sample dilution)/300
It should be noted that the seed correction for each sample dilution must be calculated when the seed is
added to the dilution water
...

DETERMINATION OF SAMPLE BOD
The calculated seed correction is subtracted from the DO depletion in the determination of the BOD for
each valid sample dilution
...
Those
sample dilutions meeting these criteria should yield the most valid results
...


The sample dilutions should deplete at least 2
...


2
...
0 mg/L after five days incubation at 20°C
...
If more than one sample dilution meets the criteria, the final BOD should be an average of

Chapter 5 - 15

the individual BOD results for the sample dilutions
...

NOTE: If this is the case, a notation should be made on the sample bench sheet that potentially invalid
data has been used to determine the noted value
...

Calculate the seeded sample BOD using formula #5 below
...
The dilution range, initial DO, final DO, and depletions (using formula #1) are given in
Table 2
...
95
7
...
90
7
...
20
3
...
40
1
...
75
4
...
50
6
...
Use formula #2 to determine the BOD of each seed dilution, then
calculate the average seed BOD
...
95 - 5
...
75 x 300)/3 = 275
Bottle #2 BOD = [(7
...
85) x 300]/6 = (4
...
90 - 2
...
50 x 300)/9 = 183
Bottle #4 BOD = [(7
...
35) x 300]/12 = (6
...
In other words, a 300 mL
sample of the undiluted seed material would use 206 mg/L DO if incubated at 20°C for five days
(assuming that oxygen was available to the sample)
...
Sample series
“A” was prepared by adding 1 mL of the seed material directly to each 300 mL sample BOD bottle
...
An unseeded dilution
water blank was also run with the series (depletion = 0
...


Chapter 5 - 16

Series “A”
Bottle #
5
6
7
8

mL Seed
10
50
75
100

Initial DO

Final DO

Depletion

8
...
95
7
...
55

6
...
60
3
...
90

1
...
35
3
...
65

Examination of the data reveals that bottle #5 can be discarded because it does not meet the 2
...

The seed correction then will only be applied to bottles #6 and #7
...
C
...
C
...
C
...
69 mg/L
Using formula #5, the BOD for each valid sample dilution can be calculated:
BOD for bottle #6 = [(3
...
69) x 300]/50 = (2
...
96 mg/L
BOD for bottle #7 = [(3
...
69) x 300]/75 = (3
...
44 mg/L
The average BOD for two valid dilutions representing the sample BOD is 14
...

Series “B”
Bottle #
9
10
11
12

mL Seed
10
50
75
100

Initial DO

Final DO

Depletion

8
...
95
7
...
15

6
...
50
3
...
70

1
...
45
3
...
45

As in series “A”, examination of the data reveals that bottles #9 and #12 can be discarded because they
do not meet both of the criteria for most valid results
...

Using formula #3, the volume (mL) of seed material in each valid dilution is determined:
mL seed in bottle #10 = (4 x 250)/1000 = 1
...
9 mL
Using formula #4, the seed correction (S
...
) for each valid dilution is determined:
S
...
for bottle #10 = (206 x 1)/300 = 0
...
C
...
9)/300 = 0
...
45 - 0
...
76 x 300)/50 = 16
...
80 - 0
...
18 x 300)/75 = 12
...
64 mg/L
...
5
1
...


What materials can be used to seed a BOD sample?

3
...
Quality Assurance (QA) is
a set of operating principles that must be followed during sample collection and analysis
...

Quality Control (QC) includes any testing which is done to prove that the results are reliable
...
This may include duplicate samples, spike
samples, reagent blank analyses and known QC samples obtained from outside sources
...
The
closer the results, the more accurate the analysis
...
Spike
sample analysis involves adding known amounts of analyte to a sample and calculating the percent
recovery
...

In BOD testing, dilution water blanks must be run with each group of samples and should not show a
depletion of more than 0
...
Duplicate samples should be analyzed to test for variability
...
Six mL of this solution in a 300 mL BOD bottle should yield 200 +/37 mg/L BOD after five days incubation at 20°C
...
A sample bench sheet is included in
Appendix C
...
1
1
...


2
...


3
...
The maximum
holding time is 48 hours at 4°C
...
2
1
...

b
...

d
...

f
...

h
...


What equipment, apparatus, or instrumentation is required for the BOD5 and/or CBOD5 test?
a
...

c
...

e
...


3
...
2;
magnesium sulfate solution;
calcium chloride solution;
ferric chloride solution;
distilled water;
glucose-glutamic acid solution; and,
nitrification inhibitor for CBOD
...


If the expected BOD of a sample is in the range 25 mg/L to 75 mg/L, what would be the minimum
and maximum sample volumes to use for the sample dilutions?
8 mL and 84 mL

Chapter 5 - 19

Quiz 5
...


Why must samples containing caustic alkalinity or acidity be adjusted before preparing BOD
dilutions?
Caustic alkalinity and acidity can prevent the growth of bacteria during the test which
prevents the use of oxygen
...


What reagents and equipment are required to adjust the pH of BOD samples?
a
...

c
...

e
...


Sodium hydroxide, 1 N;
sulfuric acid, 1 N;
pH meter;
100 and 1000 mL beakers; and,
10 mL measuring pipettes
...


4
...
0250 N; Potassium iodide solution, 10%; acetic acid (1+1) or
sulfuric acid (1+50); Starch indicator
...


What must be done to samples which have been dechlorinated or adjusted for pH variations?
They must be seeded and a seed correction used in the calculation of the BOD
...
4
1
...


2
...


What is being measured when nitrification inhibition is used in the BOD test?
Carbonaceous BOD (CBOD)

4
...
0 mg/L after 5 days and have at least 1
...


Quiz 5
...


Why must samples which have been dechlorinated or adjusted for pH variations be seeded?
Samples must be seeded to add healthy organisms to a sample which has been stressed by
toxic conditions (chlorine, high or low pH, etc
...


What materials can be used to seed a BOD sample?
Any material which can provide a suitable population of organisms can be used, however,
settled raw sewage or commercially prepared seed material are the most common sources
...


What is the acceptable range for seed correction factors in the BOD test?
0
...
4 mg/L seed correction

Chapter 5 - 21

APPENDIX A
References
Standard Methods for the Examination of Water and Wastewater, APHA-AWWA-WEF, 18™ Edition,
1992, Method 5210
...
S
...
1
...
Before working with any chemical, consult the appropriate
Material Safety Data Sheet (MSDS) to determine if other safety precautions are necessary
...
5 g potassium dihydrogen phosphate (KH2PO4), 21
...
4 g disodium hydrogen phosphate heptahydrate (Na2HPO4 7H2O), and 1
...
The pH of this buffer should be 7
...
Discard this reagent if there is any sign of biological growth in
the storage bottle
...
5 g magnesium sulfate heptahydrate (MgSO4 7H2O) in distilled water and dilute to 1 liter
...

Calcium chloride solution
Dissolve 27
...
Discard this
reagent if there is any sign of biological growth in the storage bottle
...
25 g ferric chloride hexahydrate (FeCl3 6H2O) in distilled water and dilute to 1 liter
...

Sodium hydroxide solution, 1 N
Dissolve 40 g solid sodium hydroxide (NaOH) in approximately 800 mL of carbon dioxide (CO 2) free
distilled water
...

SAFETY NOTE: This reagent is corrosive and can burn hands and clothing
...

Sulfuric acid solution, 1 N
Cautiously add 28 mL of concentrated sulfuric acid (H2SO4), with mixing, to 800 mL of distilled water
...

SAFETY NOTE: This reagent is corrosive and can burn hands and clothing
...

Sodium sulfite solution, 0
...
575 g anhydrous sodium sulfite (Na2SO3) in distilled water and dilute to 1 liter
...

Potassium iodide solution, 10%
Dissolve 10 g potassium iodide (KI) in 100 mL of distilled water
...

Acetic acid solution, 1+1
Carefully pour 50 mL of glacial acetic acid (CH3COOH) into 50 mL distilled water with mixing
...
Rinse affected areas
with large quantities of tap water to prevent injury and remove contaminated clothing, as residual
may still damage skin
...

SAFETY NOTE: This reagent is corrosive and can burn hands and clothing
...

Glucose-glutamic acid solution
Dry reagent grade glucose and reagent grade glutamic acid at 103°C for 1 hour
...
Prepare this solution fresh immediately before
use
...


Chapter 5 / Appendix B / Page 2

APPENDIX C
Sample Bench Sheet (may be duplicated for use)

BOD Benchsheet

CBOD5

BOD5

Facility Name:

Lab Tech:

Lab Tech:

Start Date:

End Date:

Time:

Time:

Method:

Method:

Sample Location
Bottle #
Initial DO
- Final DO
Depletion
- Seed Correction
= Corrected Depletion
X 300 mL
Div by Sample
Volume (mls)
BOD Result

Chapter 5 / Appendix C / Page 1



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